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The optics of microscopy

Article in Journal of Optics A Pure and Applied Optics · May 2007

DOI: 10.1088/1464-4258/9/6/S01

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IOP PUBLISHING JOURNAL OF OPTICS A: PURE AND APPLIED OPTICS
J. Opt. A: Pure Appl. Opt. 9 (2007) S1–S6 doi:10.1088/1464-4258/9/6/S01

REVIEW ARTICLE

The optics of microscopy

Colin J R Sheppard
Division of Bioengineering, National University of Singapore, 117576, Singapore
and
Department of Diagnostic Radiology, National University of Singapore, 119074, Singapore

Received 18 January 2007, accepted for publication 27 March 2007

Published 21 May 2007
Online at stacks.iop.org/JOptA/9/S1
Abstract
The optical microscope is a standard optical instrument that is used as an
example to introduce the principles of optics. However, some of the
assumptions that are used to describe image formation are highly suspect.
Take the paraxial approximation, for example, which assumes angles are
small when in practice they can be greater than 70◦ . Research into new
microscope techniques and improvements in understanding quantitatively the
image formation process has led to investigations in various areas of optics
including diffraction theory, pupil filters, non-paraxial focusing, vectorial
effects, finite Fresnel number, partial coherence, three-dimensional image
formation, beam propagation, ultra-short pulse propagation and scattering
theory. Some of these topics are reviewed, with particular emphasis on their
applications in microscopy. Also, in addition to their intrinsic scientific
interest, some of these areas have other applications, in optical information
storage and lithography for example.
This paper is based on a plenary presentation at Photonics06, Manchester
UK, on the award of the Institute of Physics Optics and Photonics Division
Prize 2006.

Keywords: microscopy, image formation, diffraction

1. Introduction numerical aperture (NA) objective can be greater than 70◦ ,

which is certainly not small.
Every elementary optics course or text includes as a (3) The scalar approximation. Many objects display
topic the optical microscope. The microscope represents birefringent properties. Polarization considerations are
an important class of instruments, and has important particularly important with high numerical aperture optics.
and widespread applications ranging through metallography, (4) The 2D planar object. Objects are often assumed to be of
geology, semiconductor devices, biology and medicine. But 2D planar geometry, but real objects are invariably three-
the elementary treatment of the microscope is based on a series dimensional. Often we are concerned with generating a
of simplifying assumptions, which may be far from justified. 3D image of a 3D object.
The classical assumptions that are made in microscope theory (5) The thin screen approximation. If the object is thick,
are: then the assumption that the complex amplitude of the
wave exiting from the specimen is given simply by a
(1) The Debye approximation. The Fresnel number of an multiplicative object transmission function is not justified.
optical system can be finite, but is usually assumed to Instead imaging can be modelled based on grating or
be infinite, which is the basis for the Debye theory of scattering theory.
focusing. (6) Incoherent or coherent image formation. Rayleigh
(2) The paraxial approximation. Elementary treatments of introduced his resolution criterion based on an assumption
geometrical and physical optics are based on a small-angle of perfectly incoherent image formation, as in the
approximation, but the angle of convergence for a high astronomical imaging of stars. In contrast, Abbe’s theory

1464-4258/07/060001+06$30.00 © 2007 IOP Publishing Ltd Printed in the UK S1

Review Article

of microscope image formation was based on a purely because the Fraunhofer diffraction region of the aperture
coherent theory. In practice image formation is often is imaged into the focal plane of the lens, the Debye
partially coherent. approximation is exactly satisfied. Normal microscope
(7) Monochromatic illumination. Elementary treatments objectives are designed to satisfy this property, but in practice
are usually based on a monochromatic (or quasi- because high numerical aperture lenses contain a lot of glass,
monochromatic) theory. Colour is sometimes an important it may not be practically feasible to place the aperture stop
property of microscope images, and different imaging exactly at the front focal plane, although the Fresnel number
methods can rely on broadband illumination, or the use is likely to be very large in most practical examples.
of ultrashort pulses, as are often used in multiphoton
microscopy. 3. The paraxial approximation
The study of optics made huge advances in the 1960s, Of much more importance in microscopy is the assumption
partly with impetus gained from the invention of the laser. of the paraxial approximation, which is far from justifiable.
An excellent textbook was published by Martin describing the Richards and Wolf described a vectorial large-angle general-
effects of these advances on our understanding of the operation ization of the Debye approximation [11]. In fact, some of
of the microscope [1]. But that book is now very out-of- these results were given many years earlier by Ignatowsky [12].
date and modern concepts such as confocal microscopy are There are three distinct generalizations that must be introduced
completely omitted. Since then, although there have been for a high NA system. First it is no longer allowable to assume
many books written for the user of the microscope, and several that angles are small so that sin θ ≈ tan θ ≈ θ . Second is
books devoted to the more modern techniques such as confocal the apodization effect that is introduced for an aplanatic sys-
microscopy, there is still not a book that effectively replaces tem (or otherwise) to satisfy conservation of energy. Third is
that of Martin. Mention should be made, however, of the book the vectorial effect on the polarization of light in traversing the
series by Pluta, that describes in detail the different specialized lens. A model of a high NA focusing system is shown in fig-
techniques, but without presenting the basic underlying theory ure 3. The lens is modelled as a black box, which converts a
in detail [2]. collimated beam into a focused beam. The intersection of rays
entering and leaving the black box defines a surface called the
2. The Debye approximation equivalent refractive locus [13], which is a sphere for a system
that satisfies the sine condition. The sine condition must be
Born and Wolf discuss the three-dimensional light distribution satisfied in order to achieve aberration-free imaging across a
near focus of a lens [3]. They consider a spherical wave finite field of view. If the sine condition is satisfied, a uniform
emerging from a circular aperture and converging towards the plane wave entering the system has an angular amplitude vari-
axial focal point. They apply the Huygens–Fresnel principle, ation cos1/2 θ after focusing, in order to satisfy conservation
and assume the distance of the observation point from the of energy [14]. A scalar high NA theory of focusing was pre-
focus is small compared with the radius of curvature of the sented by Sheppard and Matthews [15]. Interestingly, it seems
wavefront leaving the aperture, which is equivalent to the that introducing the high NA properties while retaining a scalar
Debye approximation. Under this condition the diffraction theory, overemphasizes the high NA effects.
integral reduces to an integral over the angular spectrum of We might wonder what will be the result if neither the
plane waves specified by the aperture. For an aberration-free Debye approximation nor the paraxial approximation is valid.
lens, the focused intensity is found to be symmetrical about This case is of limited applicability, as the radius of the
the focal plane. Several papers described how this symmetry pupil, and focal length, are then necessarily only several
is broken if the condition is not satisfied [4–8]. Then the wavelengths in size, but could be important in micro-optics
intensity in the focal region is distorted, so that in particular or diffractive optics. We find that the degree of focal shift
the maximum intensity is no longer achieved at the focal point, for a given value of Fresnel number also depends on the
the focal shift effect. It was shown that the condition is satisfied numerical aperture [16]. In this case we find that the predicted
if the Fresnel number N = a 2 /λ f  1 [9], so that breakdown results are different according to whether we assume Kirchhoff
of the Debye approximation is easily achieved if a/ f  1. or Rayleigh–Sommerfeld diffraction integrals, which tends to
However, even if a/ f is not small it is possible for N to give rise to concerns that a rigorous diffraction theory is really
not be very large, in areas such as micro-optics or diffractive necessary [17]. But one useful result of this theory is that it
optics for example. Sheppard [10] has described how a large predicts an axial dimensionless optical coordinate that reduces
Fresnel number is a necessary, but not sufficient, condition for to the known expressions for either finite-Fresnel number or
the Debye approximation to be valid: there is an additional paraxial systems.
condition that the distance from the focal plane must also be
small, δz  f . For an aberration-free lens, the intensity is only 4. The scalar approximation
appreciable in the focal region, but for apodized systems or in
the presence of aberrations the intensity can be appreciable far For plane polarized illumination, the polarization of the light
from the focal plane, and then the Debye approximation may is also altered during focusing, resulting in longitudinal and
not be valid even if the Fresnel number is large. cross-components of polarization being introduced [18]. The
In microscopy, there is another case when the Debye polarization of the converging spherical wave consists of
approximation is valid. This is when the aperture stop is electric and magnetic field vectors that lie on two mutually
situated in the front focal plane of the focusing lens. Then orthogonal sets of circles on the Gaussian reference sphere

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(a) (b)

Figure 2. The normalized ratio Fe of the time-averaged electric

(c)
energy density at the focus to the total power, as a function of the
angular aperture α for different polarization distributions. For small
α , the transverse electric dipole p and mixed dipole p + m exhibit
similar performance. For α → π , for the mixed dipole case the
power is divided equally between electric and magnetic energy
densities, but for the electric dipole case is concentrated in the
electric energy density. For an axially oriented electric dipole pz ,
corresponding to radially polarized illumination, the electric energy
density at the focus is small for small α , as a consequence of the
strong side-lobes in the focused spot, but for α approaching π/2
becomes greater than for the mixed dipole case. For an aplanatic
system illuminated by a plane-polarized wave, the ratio Fe is similar,
but slightly less than that for the mixed dipole case [20]. The
transverse electric dipole case p was plotted incorrectly in [21], but
Figure 1. The polarization of the electric (dashed lines) and the correct expressions were given.
magnetic (solid lines) fields on the Gaussian reference sphere for
different cases: (a) an electric dipole at the origin O oriented along
the x axis, (b) a magnetic dipole oriented along the y axis, (c) a
mixed dipole field, corresponding to an electric dipole oriented along
the x axis and a magnetic dipole oriented along the y axis. The point
C corresponds to the point on the reference sphere on the axis at the
centre of the pupils. The point A corresponds to the point on the axis
in the direction of propagation. Near to the point A the electric field
is vertical and approximately planar in each case. Radially polarized
illumination corresponds to an electric dipole oriented along the z
axis, so that the electric field lines are great circles through the
point A.

(figure 1) [19]. This polarization is identical to that of an

electric dipole along the x axis and a magnetic dipole along
the y axis placed at the focus, called a mixed dipole wave. It
is found that the electric energy density at the focus is then
maximized for a given power input if the amplitude distribution
((1 + cos θ )/2) also matches that of this mixed dipole wave Figure 3. The model for an infinity tube length lens of high
(figure 2) [20]. For the mixed dipole wave the energy excites numerical aperture, which focuses a plane wave into a converging
electric and magnetic dipoles at the focus. However usually wave. The lens is treated as a black box, and described by the
equivalent refractive locus, the intersection of the incident parallel
we are only interested in the electric field at the focus, which is
rays and the resultant converging rays. The aperture stop is placed in
maximized if the polarization of the incident light is modified the front focal plane, situated a distance f before the principal plane.
so that after focusing it matches that of an electric dipole. This
has been called an electric dipole wave (figure 1) [21]. For the
limiting case of a lens with aperture α = 90◦ , compared with
about the same width as for the Airy disc. However, using
the aplanatic case, the mixed dipole field gives an increase in
radially polarized illumination with an annular pupil results in
electric energy density at the focus of 2.3%, while the electric
dipole wave gives an increase of 17.1% (figure 2). Another a focused spot very little larger than that predicted by paraxial
way of maximizing the electric energy density at the focus is by optics [25].
matching the polarization to that of an electric dipole oriented The previous paragraphs were concerned with focusing of
along the longitudinal direction. This corresponds to radially light, rather than imaging. Effects of high NA on imaging have
polarized illumination of the lens [21–24]. For plane polarized been investigated for a scalar system [26]. It is well known that
illumination and a narrow annular pupil, the focused wave is high NA lenses that satisfy the sine condition are corrected only
equivalent to an electromagnetic Bessel beam [19]. It is found for a specific pair of conjugates. A change in the axial position
that the focal spot splits into two for high NAs, as a result of in image space introduces spherical aberration in the form of
the longitudinal electric field component at the focus [19]. As tube length error. Thus for a point object in the low NA space,
a result, for circularly polarized illumination the focal spot is the 3D variation in intensity in the high NA space is different

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from the variation in the low NA space for a point object in the illumination and the collection pupils. We have to differentiate
high NA space [26]. The principle of reciprocity still holds, between a conventional system using an incoherent source or
however. Another result is that the longitudinal magnification detector, or a confocal system where source and detector are
is not M 2 , as it is for a paraxial system. An aberration- coherent [33]. But the rigorous theories are computationally
free image cannot be formed of a 3D object in 3D space. intensive, bearing in mind that angles of incidence are high
However, using a system that satisfies the sine condition, a and structures thick, and that we must integrate over angles
series of aberration-free 2D images can be recorded (neglecting of incidence and scattering, so that the inverse problem of
aberrations caused by refractive index variations in the object) determining the object structure from the image information
by bringing successive planes of the object into the focal plane is in general intractable, hence the need for an approximate
of the system. In this way an aberration-free 3D image can be theory. For a surface structure consisting of smooth or rough
recorded and stored in a computer. surfaces, and perhaps interfaces, an approximate theory can be
A modern microscope consists of an objective used in based on reflection from these surface elements, taking into
infinity tube length configuration in conjunction with a tube account defocus effects [34] and also the optical sectioning
lens. Thus the complete system is modelled by combining property of a confocal system [35]. This is analogous to the
the objective as modelled in figure 3 together with a low NA Kirchhoff approximation in scattering theory [36]. It turns out
tube lens [26]. It is rather convenient that microscope objective that the object can now be completely specified as a function
manufacturers have adopted the infinity tube length system, as of three spatial frequencies, rather than the four direction
a finite tube length objective, as was universal until recently, is cosines needed to specify scattering for arbitrary directions of
more difficult to understand conceptually [27, 28]. illumination and scattering. This suggests that the 3D spatial
frequency model can provide insight into the Kirchhoff theory
5. The 2D planar object of scattering [37]. This approach can be generalized to include
scattering from refractive index variations inside a bulk 3D
In conventional imaging theory, we are concerned with object [38, 39].
imaging of a planar object. How can we extend this approach
to encompass thick 3D structures? Wolf showed that for a 7. Incoherent or coherent image formation
weakly scattering object, so that the first Born approximation
is valid, each successive plane of the object is imaged Abbe introduced the Fourier theory of both coherent and
independently [29]. Then if the spatial frequency content of incoherent image formation [40]. But it was not until Hopkins’
the 3D object is considered, it is found that in a coherent seminal paper that image formation in a partially coherent
imaging system only grating vectors that lie on the surface of a system was fully understood [41]. When the present author
spherical shell passing thorough the origin of Fourier space are tried to publish a theory of imaging in confocal microscopes,
imaged. This is because both the incident and scattered wave he experienced difficulty in getting it accepted, mainly because
vectors must have equal magnitude. This concept for imaging, the accepted view at that time was that Hopkins’ theory
related to the Ewald sphere construction of x-ray diffraction, is encompassed all forms of microscope imaging systems. Now
analogous to one for focusing introduced by McCutchen [30]. we appreciate that Hopkins’ theory is restricted to systems
He showed that the amplitude in the focal region of a lens that use an extended incoherent source, resulting in partially
is given very simply as the 3D Fourier transform of the cap coherent illumination of the object. This theory was extended
of a sphere representing a spherical wavefront, the 3D pupil to the case of coherent sources [42], then to partially coherent
function. Frieden [31] and Mertz [32] considered incoherent sources (and detectors) [43], and then to systems with
imaging, so that the resultant optical transfer function is given structured illumination [44, 45].
by the autocorrelation of the pupil function, in this case in 3D Hopkins presented the defocused optical transfer function
rather than in 2D as is conventionally the case. For incoherent for an incoherent microscope [46], but it was not until recently
imaging, now a continuous region of Fourier space is imaged, that the defocused transfer function for a weak object in a
corresponding to the support for spatial frequencies present in partially coherent microscope has been presented [47].
the image, but there is a missing cone of spatial frequencies, Of course fluorescence microscopes always behave
representing detail that has low transverse spatial frequency incoherently, but still we must appreciate that we can employ
coupled with high longitudinal spatial frequency, that are not coherent illumination systems of different geometries to excite
imaged. This constitutes the limitation of conventional optical fluorescence [48–50], thus resulting in improved imaging
systems for imaging of 3D object structures. performance. For confocal or structured illumination systems,
the spatial frequency bandwidth can be extended by a factor of
6. The thin screen approximation two relative to conventional incoherent systems.

The first Born approximation was introduced for imaging in a 8. Monochromatic illumination
transmission geometry. But basically the image of any thick
structure in transmission or reflection can be calculated by Image formation is often described in terms of monochromatic
considering scattering by the object. Each plane wave of illumination, but in practice broadband CW illumination
the illumination results in an angular spectrum of scattered is often used. In some modes of modern microscopy
radiation, the strength of which can be calculated by rigorous (e.g. multiphoton microscopy) ultra-short pulses, equivalent to
diffraction theory (modal, coupled wave or integral equation a spread of frequency, are used. These two cases correspond to
theories). Then the scattered radiation is summed over both the incoherent or coherent summation of the spectral components,

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Figure 4. Spectral components of polychromatic beams: (a) beams of type 1 (same waist), (b) type 2 (same angular spectrum) and (c) type 3
(isodiffracting). The blue components are shown as dashed lines and the red components as solid lines.
(This figure is in colour only in the electronic version)

respectively. It is interesting to note that the intermediate case in the traditional theory. In particular, we have discussed
of partially coherent summation, often considered for spatial effects of finite Fresnel number (breakdown of the Debye
coherence, is rarely considered for the temporal case. Of approximation), high NA focusing and imaging, effects of
course the fact that different wavelengths result in different polarization, and the interaction with a thick 3D object.
resolutions is obvious, and must have been well known Although the discussion has been necessarily brief, it is hoped
to Rayleigh and Abbe. A focused spot produced by a that the selected references will allow the interested reader to
chromatically corrected lens has a diameter proportional to study these topics in greater detail. There are still some areas
the wavenumber. As a result, the spectral distribution at that have yet to be investigated in depth. These include the
different points in the focal region inevitably varies, being effects of polarization in 3D imaging of thick objects and 3D
in general blueshifted near the axis and redshifted far from partially coherent imaging.
the axis. In the vicinity of a zero of the Airy disc at the
central wavelength we would expect the spectral distribution References
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